Summary/Abstract Trisomy 21 (T21) is the most common human chromosomal anomaly, affecting 1 in 700 live births, resulting in Down Syndrome (DS). Although DS can affect many organ systems, lung and heart disease are the leading causes of morbidity and mortality. Several congenital lung anomalies are reported in individuals with DS including airway branching defects, with a 25% decrease in the number of branches and reduced upper airway muscle tone with dysphagia and/or bronchomalacia. These complications remain constant into adulthood, as opposed to becoming exacerbated, and are hence likely due to developmental insufficiency. While abnormal pulmonary structure and function in pediatric and adult individuals with DS has been described, there is limited data defining the ontogeny of these abnormalities. We postulated that developmental differences could initiate prenatally in T21 lungs and have now demonstrated pulmonary anomalies arise in approximately 70% of T21 cases starting as early as 16 weeks gestation, as determined from experiments proposed in the parent grant. Our single cell RNAseq data and immunofluorescent stainings have demonstrated upregulation of the surfactant metabolism pathway and increased cell number of differentiated proximal airway markers (SCGB1A1 and FOXJ1) respectively. Altogether these findings strongly support one of the hypotheses proposed in the parent grant, where we postulate precocious development/differentiation in the T21 lungs as compared to their age and sex matched controls. These observations are fitting being that DS is generally described as a progeroid syndrome. It has been demonstrated that this accelerated aging may be associated to genome wide perturbation due to dysregulated epigenetic regulation; however, no studies have investigated the epigenetic landscape of the developing T21 lung. Thus, we hypothesize cellular and molecular abnormalities in T21 lungs initiate in utero, resulting in precocious development associated with altered epigenetic regulation. Recent advances in single cell epigenetic profiling methodology allow us to simultaneously test this hypothesis in the same tissues we are collecting and analyzing for the parent project. We will use multiome sequencing (single nuclear RNAseq/ATACseq) to determine the cellular and molecular abnormalities initiated during the late pseudoglandular/early canalicular stages of fetal development in T21 lungs. Being that epigenetic modifications are preserved in daughter cells, we will also determine alterations in gene regulatory networks at the single cell level between prenatal and postnatal T21 lungs vs non-T21 lungs, to further define the ontogeny of pulmonary anomalies observed in DS. These studies have the promise to improve our understanding of the key molecular and cellular differences, and the mechanisms controlling defects in T21 developing lungs. The innovative aspects of this work are likely to have great overall impact in stimulating additional activity leading to progress on understanding lung anomalies in DS and facilitate the translational potential for therapies.

Project Narrative Trisomy 21 (T21) is the most prevalent chromosomal abnormality worldwide, with an incidence of approximately 1 in 700 live births. Our single cell RNAseq data and immunofluorescent stainings have demonstrated upregulation of the surfactant metabolism pathway and increased cell number of differentiated proximal airway markers (SCGB1A1 and FOXJ1), suggesting precocious development/differentiation in the T21 lungs, which may be associated to genome wide perturbation due to dysregulated epigenetic regulation. In this proposal we will assess the ontogeny of pulmonary anomalies observed in Down Syndrome (DS) by using single nuclear (sn) RNA and snATAC sequencing (multiome sequencing) to simultaneously assess the epigenetic landscape and determine the cellular and molecular abnormalities observed in developing T21 lungs (from prenatal to postnatal), which may ultimately provide a foundation for potentially new therapies for compromised lungs in individuals with DS.